1. Field of the Invention
The present invention relates to electrospray ionization mass spectrometry, and more particularly to a method of charge reduction whereby ions produced by electrospray are amenable to partial neutralization and subsequent detection by an orthogonal time-of-flight mass spectrometer to yield high resolution mixture spectra.
2. Description of Related Art
The structure of deoxyribonucleic acid (DNA) consists of two parallel strands connected by hydrogen bonding. Double stranded DNA molecules assume a double helix structure with varying geometric characteristics. Under certain salt or temperature conditions, denaturation can occur and the two DNA strands become separated.
The order of nucleotides along a single strand corresponds to the sequence of DNA. Each set of three contiguous bases (a codon) encodes a particular amino acid used in protein synthesis. Successive codons are organized into a gene to encode a particular protein. DNA is thus present in living cells as the fundamental genetic information carrier.
The human genome is the complete set of human DNA present in every cell (apart from reproductive and red blood cells). It is believed that total human DNA comprises 3 billion base pairs encoding about 100,000 genes. Sequencing the entire genome is desirable because knowledge of gene sequencing should increase the understanding of gene regulation and function and allow precise diagnostics and treatment of genetic diseases.
Using current sequencing technologies, about 14,000 base pairs can be acquired in 14 hours in an electrophoresis gel. The ultimate goal of 3 billion base pairs therefore poses a technological challenge and presents a need for high performance sequencing instruments. To this end, mass spectrometry can be used as a sequencing technique.
An important field emerging from genomics is proteomics. Proteomics concerns the study of all the proteins encoded for by genes. Like genomics, proteomics involves extremely complex mixtures of large biopolymers (proteins in this case) that need to be separated and identified. Current technologies mainly make use of 2-D electrophoresis gels, which separate proteins based on both size and the isolelectric point of the proteins. These gels are labor intensive to prepare and time-consuming to run and analyze. Mass spectrometry offers a high-speed, high-sensitivity, low-labor alternative to separate, sequence, and identify complex mixtures of proteins.
Mass spectrometry allows the acquisition of molecular weights (measured in daltons) for every mass to charge (m/z) peak acquired, whereby the m/z ratio is an intrinsic and condition-independent property of an ion. By eliminating the preparation of gels required with electrophoretic mobility analysis, mass spectrometry has the potential for requiring only milliseconds per analysis. By its nature, it is an intrinsically fast and accurate means for accurately assessing molecular weights.
Mass spectrometry requires that the analyte of interest be produced in the form of a gas phase ion, within the vacuum of a mass spectrometer for analysis. While achieving this is straightforward for small molecules using classical techniques (such as sublimation or thermal desorption) used in conjunction with an ionization method (such as electron impact), it is much less straight-forward for large biopolymers with essentially nonexistent vapor pressures. For this reason, the field of large-molecule mass spectrometry was extremely limited for many years. This situation changed dramatically with the discovery of two important new techniques for producing ions of large biomolecules (macromolecules), namely Matrix Assisted Laser Desorption-lonization (MALDI) and Electrospray Ionization (ESI), whereby rapidly determining the mass of large molecules became feasible.
In MALDI mass spectrometry, a few hundred femtomoles of analyte are mixed on a probe tip with a small, organic, ultra-violet (UV) absorbing compound, the matrix. The analyte-matrix is dried to produce a heterogenous crystalline dispersion, and then irradiated with a brief (i.e., 10 ns) pulse of UV laser radiation in order to volatilize the sample and produce gas phase ions of the analyte amenable to mass spectrometric analysis. Because the UV pulse is at a wavelength that is absorbed by the matrix and not the analyte, the matrix is vaporized, and analyte molecules become entrained in the resultant gas phase plume where they are ionized in gas phase proton transfer reactions. However, analyte fragmentation and poorly understood matrix effects occur during the MALDI process, thereby reducing molecular ion intensity and complicating the analysis and interpretation of the mass spectra. As a result, the mass range of this technique is limited; it frequently does not allow sequencing fragments longer then 35-100 base pairs in length.
Electrospray ionization mass spectrometry (ESI-MS), on the other hand, allows analysis of DNA with reduced fragmentation. ESI-MS is characterized by a gentle analyte desorption process that can leave noncovalent bonds intact. This soft ionization allows analysis of intact DNA molecular ions. However, ESI-MS typically produces multiply charged ions, and as the number of possible charge states increases with the size of the analyte, this technique yields complex spectra for large molecules. For example, while ESI analysis of simple molecules may be accomplished using computer algorithms that transform the multiply charged mass spectra to xe2x80x9czero-chargexe2x80x9d spectra, permitting easy visual interpretation thereof, as spectral complexity and chemical noise levels increase, these algorithms produce artificial peaks and miss analyte peaks with low signal intensity. Furthermore, each analyte yields a specific peak distribution and mixture spectra are therefore characterized by complex overlapping distributions for which the resultant spectra cannot be resolved without expensive high resolution mass spectrometers. This multiple charging and peak multiplicity in ESI-MS considerably limit the utility of this technique in the analysis of mixtures such as DNA sequencing ladders or complex protein mixtures, and serious efforts to utilize ESI-MS as a sequencing tool have thus been hampered by the complexity of the resultant mass spectra.
To make ESI-MS more effective, it is desirable to decrease the charge state of electrospray generated ions. Previous approaches to charge reduction in ESI have fallen into two major categories: modification of the solution conditions (i.e., buffer, pH, salts) and utilization of gas-phase reactions within an ion trap spectrometer. Altering solution conditions does not allow predictable and controllable manipulation of the charge state for all species present in a given mixture. With conventional ion trap techniques, the cation or anion used to reduce charge has to be xe2x80x9ctrappedxe2x80x9d along with the analyte(s). This has the practical consequence of limiting the charge reduction to a narrow m/z range of ions. Thus, previous ion trap apparatuses are limited by the nature of the ion trap to a defined m/z range and are thus not amenable to the charge reduction of large m/z ions. This is of course critical for reducing the charge of large DNA molecules.
As is evident from the foregoing, a need exists for a method of combining the simplicity of singly charged species spectra with the softness of ESI to efficiently and effectively allow high resolution mass spectral analysis of a mixture of a sample analyte solution containing a macromolecule of interest in a solvent wherein the method used is not limited to a low m/z range and wherein off-line sample purification or pre-separation is not required.
The method of the present invention enables mass spectral analysis of a solution containing a macromolecule of interest by preparing a sample analyte solution containing the macromolecule in a solvent, discharging, with assistance of a nebulizing gas, the analyte solution through an orifice held at a high voltage in order to produce a plurality of analyte droplets that are multiply charged, evaporating the solvent in the presence of a bath gas in order to provide a plurality of macromolecule particles having multiple charges, exposing the bath gas proximal to the macromolecule particles to a radioactive alpha-particle emitting source that ionizes elements of the bath gas into bipolar ions, controlling the interaction time between the macromolecule particles and the bipolar ions in order to reduce the multiply charged macromolecule particles to predominantly singly charged particles, and then analyzing the stream of singly charged macromolecule particles in a mass spectrometer.
More specifically, a sample analyte solution is placed into a vessel in an ESI source and discharged as an aerosol through an orifice held at a high potential. Due to a voltage differential between the spray tip orifice and the internal walls of the ESI source, an electrostatic field is created whereby charges accumulate at the surface of the emerging droplets. Charge reduction is achieved by exposure of the aerosol to a high concentration of bipolar ions (i.e., both positively and negatively charged ions present in the charge reduction chamber). Collisions between the charged aerosol and the bipolar ions in the bath gas result in the neutralization of the multiply charged electrospray ions. The rate of this process is controlled by varying the concentration of the bipolar ions in the bath gas and the degree of aerosol exposure to an ionization source such as Polonium (210Po), a radioactive metallic element that emits alpha particles to form an isotope of lead. This provides, in effect, the ability to xe2x80x9ctunexe2x80x9d the charge state of the electrospray generated ions. A practical consequence is the ability to control the charge distribution of electrospray generated ions such that the ions can be manipulated to consist principally of singly charged ions and/or douly charged ions, thereby simplifying mass spectral analysis of DNA and protein mixtures.
By the disclosed method, the present inventors have succeeded in using an ESI-TOFMS (electrospray ionization-time of flight mass spectrometry) to analyze particles ranging from 4 to 8 kDa in size. In this technique, the particles in the continuous liquid flow from the electrospray source are desorbed and ionized. The resultant multiply charged species are then neutralized by passage through a neutralizing chamber whereby singly charged macromolecules result. As a result, the charge state of the ions generated in the electrospray chamber are reduced in a controlled manner whereby the stream of singly charged macromolecules are analyzed in a mass spectrometer such as an orthogonal time-of-flight (TOF) mass spectrometer, yielding high resolution mass spectra.
The method described herein decouples the ion production process from the neutralization process. This is important because it provides flexibility with respect to the electrospray conditions, which is critical to obtaining high-quality results, and it permits control over the degree of charge neutralization. In addition, with the approach presented here, the cation or anion used to reduce charge does not have to be xe2x80x9ctrappedxe2x80x9d with the electrospray ions. This has the practical consequence of permitting the charge reduction to be performed on virtually any m/z ranges of ions, independent of the neutralizing cation or anion""s m/z value. In addition, because a specific anionic or cationic species is not required in the method of this invention, switching between positive and negative modes of electrospray is straightforward. This allows protein cations to be neutralized in positive ion mode or DNA anions to be neutralized in negative ion mode without having to change any instrumental conditions other than operating polarity.
It is thus one object of this invention to allow rapid analysis of mixtures of synthetic or naturally occurring biopolymers with high m/z ranges for a wide range of applications. It is another object of the present invention to accomplish the above objective without requiring a major change in standard operational procedures. It is yet another objective of the present invention to accomplish the above objectives with a minimal cost adjustment over traditional ESI, thereby permitting accurate, high speed, high resolution, and low cost effective mass determinations of DNA macromolecules without requiring preparation of a mixture on a column or being subject to the limitations of traditional ion traps.
In an alternative embodiment, the present invention provides methods and devices for generating ions from liquid samples containing chemical species, including but not limited to chemical species with high molecular masses. In a preferred embodiment, the ion source of the present invention comprises a flow of bath gas that conducts the output of an electrically charged droplet source through a field desorption-charge reduction region cooperatively connected to the electrically charged droplet source and positioned at a selected distance downstream with respect to the flow of bath gas. The generation of electrically charged droplets in the present invention may be performed by any means capable of generating a continuous or pulsed stream of charged droplets from liquid samples containing chemical species. In an exemplary embodiment, an electrospray ionization charged droplet source is employed. Other electrically charged droplet sources useful in the present invention include but are not limited to: nebulizers, pneumatic nebulizers, thermospray vaporizers, cylindrical capacitor generators, atomizers, and piezoelectric pneumatic nebulizers.
First, the electrically charged droplet source generates a continuous or pulsed stream of electrically charged droplets by dispersing a liquid sample containing at least one chemical species in at least one solvent, carrier liquid or both into a flow of bath gas. Chemical species refers to a collection of one or more atoms, molecules and macromolecules and includes but is not limited to polymers such as peptides, oligonucleotides, carbohydrates, polysaccharides, glycoproteins and lipids. The droplets formed may possess either positive or negative polarity corresponding to the desired polarity of ions to be generated. Next, the stream of charged droplets and bath gas is conducted through a field desorption-charge reduction region wherein solvent and/or carrier liquid is removed from the droplets by at least partial evaporation to produce a flowing stream of smaller charged droplets and multiply charged gas phase analyte ions. Evaporation of positively charged droplets results in formation of gas phase analyte ions with multiple positive charges and evaporation of negatively charged droplets results in formation of gas phase analyte ions with multiple negative charges. Gas phase analyte ions refer to multiply charged ions, singly charged ions or both generated from chemical species in liquid samples. Gas phase analyte ions are positively charged, negatively charged or both and are characterized in terms of their charge-state distribution which is selectively adjustable in the present invention. Charge-state distribution refers to a two-dimensional representation of the number of ions of a given elemental composition that populate each ionic state present in a sample of ions.
Within the field desorption-charge reduction region, the stream of charged droplets, gas phase analyte ions or both are exposed to electrons and/or gas phase reagent ions of opposite polarity generated from bath gas molecules within at least a portion of the field desorption charge reduction region by a radioactive reagent ion source. In the present invention, the radioactive reagent ion source is operationally connected to the field desorption-charge reduction region to provide a flux of ionizing radiation into the field desorption-charge reduction region. Radioactive reagent ion sources of the present invention are any means capable of providing ionizing radiation to the field desorption-charge reduction region and include but are not limited to alpha particle emitters. In the present invention, ionizing radiation refers to xcex1, xcex2, xcex3 or x-rays as well as protons, neutrons and other particles such as pions. In a preferred embodiment, the radioactive reagent ion source is a radio isotope source such as a 210Po radio isotope source or a 241Am radio isotope source. Reagent ions refer to a collection of gas phase ions of positive polarity, negative polarity or both that is generated upon ionization of bath gas molecules in at least part of the field desorption-charge reduction region by ionizing radiation generated by the radioactive reagent ion source. Optionally, reagent ions may refer to free electrons in the gas phase generated within the volume of the field desorption-charge reduction region by the flux of ionizing radiation generated by the radioactive reagent ion source. In a preferred embodiment, the reagent ions of the present invention comprise positively charged ions and negatively charge ions.
The radioactive reagent ion source is positioned at a selected distance downstream of the electrically charged droplet source and is configured in a manner to provide a source of ionizing radiation to at least a portion of the volume of the field desorption-charge reduction region. In a preferred embodiment, the flux of ionizing radiation into the field desorption-charge reduction region is selectively adjustable by use of a radiative flux attenuator element positioned between the field desorption-charge reduction region and the radioactive reagent ion source. Accordingly, the concentration and spacial distribution of reagent ions in the field desorption-charge reduction region may be selected by controlling the net flux and spacial characteristics of the output of the radioactive reagent ion source reaching the field desorption-charge reduction region. Control of the flux and spacial characteristic is provided by selectively adjusting the radiative flux attenuator element. The radiative flux attenuator element may comprise any means capable of reducing the flux of ionizing radiation into the field desorption region from the radioactive reagent ion source. In a preferred embodiment, the radiative flux attenuator element comprises at least one thin brass disc with a plurality of holes of known area drilled therein. In a more preferred embodiment, the holes drilled through the brass discs have an area of about 0.53 cm2. In another preferred embodiment, the radiative flux attenuator element comprises at least one metal screen.
The charged droplets, analyte ions or both remain in the field desorption-charge reduction region for a selected residence time or dwell time. This time is controllable by selectively adjusting the flow rate of bath gas and/or the length of the field desorption-charge reduction region. Within at least a portion of the field desorption-charge reduction region, electrons, reagent ions or both, generated by the radioactive reagent ion source, react with charged droplets, analyte ions or both to reduce the charge-state distribution of the analyte ions in the flow of bath gas. Accordingly, ion-ion, ion-droplet, electron-ion and/or electron-droplet reactions result in the formation of gas phase analyte ions having a selected charge-state distribution. In a preferred embodiment, the ion source of the present invention generates an output of gas phase analyte ions comprising substantially of singly charged ions and/or doubly charged ions.
In a preferred embodiment, the charge state distribution of gas phase analyte ions is selectively adjustable by varying the interaction time between gas phase analyte ions and/or charged droplets and gas phase reagent ions and/or electrons. This may be accomplished by varying the residence time gas phase analyte ions spend in the field desorption-charge reduction region by either adjusting the flow rate of bath gases through the field desorption-charge reduction region or by varying the length and/or physical dimensions of the field desorption-charge reduction region. Longer residence times yield greater reduction in the analyte ion charge state distribution than shorter residence times. In addition, the charge-state distribution of gas phase analyte ions may be controlled by adjusting the rate of production of electrons, reagent ions in the field desorption-charge reduction regions. This may be accomplished by either increasing or decreasing the flux of ionizing radiation into the field desorption-charge reduction region. Higher production rates of reagent ions and/or electrons yield greater reagent ion and/or electron concentrations in the field deasorption-charge reduction region. Accordingly, higher production rates of reagent ions and/or electrons in the field desorption-charge reduction region yield a greater net extent of charge reduction than lower production rates. Further, an ion source of the present invention is capable of generating an output comprising analyte ions with a charge-state distribution that may be selected or may be varied as a function of time.
Optionally, the ion source of the present invention may be operationally coupled to a device capable of classifying and detecting charged particles such as a charged particle analyzer. Charged particle analyzer refers to any devices or techniques for determining the identity, properties or abundance of charged particles. This embodiment provides a method of determining the composition and identity of substances which may be present in a mixture. In an exemplary embodiment, the ion source of the present invention is coupled to a mass analyzer and provides a method of identifying the presence of and quantifying the abundance of analytes in liquid samples. In this embodiment, the output of the ion source is drawn into a mass analyzer to determine the mass to charge ratios (m/z) of the gas phase analyte ions generated from dispersion of the liquid sample into droplets followed by subsequent charge reduction. In an exemplary embodiment, the ion source of the present invention is coupled to a time of flight mass spectrometer to provide accurate measurement of m/z for compounds with molecular masses ranging from about 1 to about 30,000 amu. Other mass analyzers useful in the present invention include, but are not limited to, quadrupole mass spectrometers, tandem mass spectrometers, ion traps or combinations of these mass analyzers.
In the ion source of the present invention, the distance between the electrically charged droplet source and the radioactive reagent ion source is selectively adjustable. In a preferred embodiment, the charged droplet source and/or the radioactive reagent ion source is moveable along a central chamber axis to permit adjustment of this dimension. It is believed that variation of this distance affects the field desorption conditions and extent of field desorption achieved. Accordingly, changing the distance between the droplet source and the radioactive reagent ion source is expected to affect the total output of the ion source of the present invention. Larger distances between the droplet source and the radioactive reagent ion source tend to allow for a greater extent of field desorption than shorter distances and, hence, tend to result in greater net ion production. In addition, variation of the distance between the droplet source and the radioactive reagent ion source also affects field desorption conditions by changing the distribution of charge at the surface of the charged droplets. A smaller distance between droplet source and radioactive reagent ion source is expected to lead to greater reagent ion-charged droplet interaction, thereby attenuating the charge on the droplet""s surface by charge scavenging. Scavenging of charge on the surface of the droplets is believed to have several effects on the field desorption process. First, charge scavenging may cause a net reduction in the extent and/or rate of field desorption of ions. Second, it may result in generation of analyte ions with a lower charge state distribution than that observed in the absence of charge scavenging. Finally, charge scavenging also tends to preserve the size distribution possessed by the electrically charged droplets upon discharge.
Alternatively, the ion source of the present invention includes embodiments comprising an electrically charged droplet source cooperatively connected to a field desorption region and a charge reduction region that are spatially separated from each other. Multiply charged droplets are generated by the electrically charged droplet source and conducted through a field desorption region by a flow of bath gas. In the separate field desorption region, solvent and/or carrier liquid is removed from the droplets by at least partial evaporation to produce a flowing stream of smaller charged droplets and multiply charged gas phase analyte ions. Evaporation of positively charged droplets results in formation of gas phase analyte ions with multiple positive charges and evaporation of negatively charged droplets results in formation of gas phase analyte ions with multiple negative charges. The charged droplets, analyte ions or both remain in the field desorption region for a selected residence time controllable by selectively adjusting the flow rate of bath gas and/or the length of the field desorption region.
Next, the stream of droplets, analyte ions or both is conducted through a separate charge reduction region operationally connected to the field desorption region and cooperatively connected to a radioactive reagent ion source. Within at least a portion of the charge reduction region, electrons, reagent ions or both, generated from bath gas molecules by ionizing radiation, react with charged droplets, analyte ions or both to reduce the charge-state distribution of the analyte ions in the flow of bath gas. Accordingly, ion-ion, ion-droplet, electron-ion and/or electron droplet reactions in the charge reduction region result in the formation of gas phase analyte ions having a selected charge-state distribution. In a preferred embodiment, the charge state distribution of gas phase analyte ions is selectively adjustable by varying the interaction time between gas phase analyte ions and/or charged droplets and the gas phase reagent ions and/or electrons.
In this alternative embodiment, field desorption and charge reduction regions may be housed in separate chambers or may merely be separated from each other by a distance large enough to provide a field desorption region substantially free of reagent ions. Ion sources with discrete field desorption and charge reduction regions are beneficial because they decouple ion formation and neutralization processes. Accordingly, experimental conditions may be optimized in the field desorption region to obtain high yields of gas phase analyte ions and experimental conditions may be independently optimized in the charge reduction region to yield the desired extent of charge reduction. This characteristic is beneficial because it provides flexibility with respect to the electrospray and field desorption conditions employable in the present invention. This flexibility facilitates obtaining high yields of singly and/or double charged analyte ions from hard to ionize species, such as polar species that do not ionize in solution.
The foregoing and other objects, advantages, and aspects of the present invention will become apparent from the following description. In the description, reference is made to the accompanying drawings which form a part hereof, and in which there is shown, by way of illustration, a preferred embodiment of the present invention. Such embodiment does not necessarily represent the full scope of the invention, however, and reference must also be made to the claims herein for properly interpreting the scope of this invention.